Nucleic acid mismatich detection

ABSTRACT

In one aspect, the invention provides methods and apparatus for detecting a mismatch in a nucleic acid duplex by measuring the impedance of a nucleic acid layer on an electrode, for example by AC impedance spectroscopy.

FIELD OF THE INVENTION

The invention is in the field of nucleic acid chemistry, particularly electrochemical techniques for analysis of nucleic acids.

BACKGROUND OF THE INVENTION

The electronic conductivity of DNA may be utilized in the development of DNA biosensors, so called “DNA chips” (Bixon et al., 1999; Schena et al., 1996; Fodor et al., 1993). One form of DNA chip consists of single-stranded DNA probes attached to a surface in an array format. The target DNA may be labelled with a fluorescent tag and successful hybridization to an individual probe may be detected fluorometrically. Electrochemical detection, on the other hand, may allow a direct readout of the signal (Takagi, 2001; Kelly et al., 1999). Electrochemical techniques include potential step chronoamperometry, dc cyclic voltammetry, and electrochemical impedance (Bard and Faulkner, 2001). Electrochemical DNA sensors may utilize electrochemically active DNA binding drugs such as the metal coordination complex Ru(bpy)₃ ²⁺ (Carter and Bard, 1987, Millan et al., 1994), electroactive dyes (Hashimoto et al., 1994), quinones (Kertesz et al., 2000; Ambroise and Maiya, 2000), and methyl blue (Tani et al., 2001; Kelley et al., 1997) as the detection markers. In other cases the simple redox probe, Fe(CN)₆ ^(3−/4−), has been used in solution (Patolsky et al., 2001). In some of these techniques, target DNA need not be labeled in advance.

The electronic characteristics of surface modified electrodes can be probed with impedance spectroscopy and the data modeled by an equivalent circuit (Macdonald, 1987). Alternative methods of electrochemical impedance spectroscopy are for example disclosed in U.S. Pat. No. 6,556,001 (incorporated herein by reference). Electron transfer through self-assembled alkanethiol monolayers or, metal surfaces has been intensively studied in recent years (Ulman, 1996). The impedance of an electrode undergoing heterogeneous electron transfer through a self-assembled monolayer is usually described on the basis of the model developed by Randles (Randles, 1947).

Duplex DNA contains a stacked π system and the conductivity of native DNA (B-DNA) has been hotly debated. Recent direct measurements suggest that B-DNA is a semiconductor with a wide band gap (Storm et al., 2001); (Rakitin et al., 2001); (Porath et al., 2000); (Murphy et al., 1993). The conductivity of DNA can be improved by deposition of silver atoms along its length but the process is essentially irreversible (Braun et al., 1998). Another possibility is to convert B-DNA to M-DNA by the addition of divalent metal ions (Zn²⁺, Co²⁺ and Ni²⁺) at pHs above 8.5 (Lee et al., 1993) (Aich et al., 1999). In M-DNA, it is proposed that the metal ions replace the imino protons of guanine and thymine in every base pair but the structure can be converted back to B-DNA by chelating the metal ions with EDTA or reducing the pH. Electron transport through M-DNA can be monitored by fluorescence spectroscopy of duplexes labelled at opposite ends with donor and acceptor chromophores. Upon formation of M-DNA the donor is quenched but only when the acceptor is on the same DNA molecule (Aich et al., 1999; Aich et al., 2002). Recent direct measurements have confirmed that M-DNA shows metallic-like conductivity and electron transfer can be observed in duplexes as long as 500 base pairs (Rakitin et al., 2001). Therefore, M-DNA may be useful in biosensor applications by allowing a direct electronic readout of the state of the DNA.

SUMMARY OF THE INVENTION

In various aspects, the invention provides methods and apparatus for electrochemical nucleic acid analysis.

In one aspect, the invention provides hardware and software for an impedance spectroscopy system that characterizes polymers such as nucleic acids by measuring impedance at various frequencies. The hardware may for example provide voltage and current Inputs to a sample at various frequencies and measure the resulting impedance. The software may store equivalent circuit parameters for multiple samples, control the hardware inputs to the sample, display measurement data, display results, and notify an operator if results exceed preset limits.

In various aspects, the invention provides methods for detecting base pair mismatches in a nucleic acid duplex tethered to an electrode in an electrochemical circuit. A plurality of nucleic acids may for example form a monolayer of nucleic acid duplexes on the electrode. The nucleic acids may be comprised of naturally occurring monomers, such as DNA and RNA, or may have synthetic substituents comprised of a wide range of alternative monomeric units.

Methods of the invention may include the steps of: a) applying electrical energy to the electrode in the electrochemical circuit; b) collecting electrochemical circuit data related to the impedance of the nucleic acid duplex on the electrode in the circuit; and, c) fitting the electrochemical circuit data to a circuit model to obtain circuit performance information indicative of a base pair mismatch in the nucleic acid duplex.

In alternative aspects, the invention provides systems for detecting base pair mismatches. Such systems may for example include: a) means such as an electrical current source for applying electrical energy to the electrode in the electrochemical circuit; b) means such as a controller for collecting electrochemical circuit data related to the impedance of the nucleic acid duplex on the electrode in the circuit; and, c) means such as an analyzer for fitting the electrochemical circuit data to a circuit model to obtain circuit performance information indicative of a base pair mismatch in the nucleic acid duplex. Such systems may further comprise a display or means for displaying the circuit performance information; and/or a recorder or means for recording the circuit performance information. The circuit performance information may for example be plotted on a Nyquist plot.

In alternative embodiments, collecting electrochemical circuit data may include measuring impedance spectra, such as impedance spectra measured in the frequency domain. Various electrochemical circuit parameters provide data that is related to the impedance of the nucleic acid duplex. For example, the real and imaginary impedance of a nucleic acid or monolayer is related to electrochemical parameters such as the Warburg impedance, the capacitance of the monolayer, the charge transfer resistance and the rate of electron transfer. Such parameters may also be used to distinguish a mismatch DNA sample from a fully duplex DNA sample.

The electrochemical circuit data of the invention may include a measure of complex impedance. In some embodiments, electrical energy may be applied in an impedance spectroscopy system, and the impedance spectroscopy system may involve applying a sinusoidal signal at a constant frequency and a constant amplitude within a discrete period. In selected embodiments, the circuit model may include circuit elements, such as:

a solution resistance Rs;

a charge transfer resistance RCT;

a constant-phase element CPE;

a mass transfer element W (Warburg impedance); and,

a resistance in parallel Rx;

wherein the circuit elements are arranged as illustrated in FIG. 1.

In some embodiments, the nucleic acid may be a deoxyribonucleic acid (DNA), and the nucleic acid duplex may be an double helix. In some embodiments, the nucleic acid may comprise M-DNA, a metal-containing nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, the first and the second nucleic acid strands comprising a plurality of nitrogen-containing aromatic bases covalently linked by a backbone, the nitrogen-containing aromatic bases of the first nucleic acid strand being joined by hydrogen bonding to the nitrogen-containing aromatic bases of the second nucleic acid strand, the nitrogen-containing aromatic bases on the first and the second nucleic acid strands forming hydrogen-bonded base pairs in stacked arrangement along the length of the conductive metal-containing nucleic acid duplex, the hydrogen-bonded base pairs comprising an interchelated divalent metal cation coordinated to a nitrogen atom in one of the aromatic nitrogen-containing aromatic bases.

The invention may involve comparing the circuit performance information of a first nucleic acid duplex to the circuit performance information of a second nucleic acid duplex. For example, the first nucleic acid duplex may be a B-DNA and the second nucleic acid duplex may be a metal-containing nucleic acid duplex, M-DNA.

The electrochemical circuit may for example include an aqueous electrolyte and the nucleic acid may be tethered and solvated in the aqueous electrolyte. A redox probe may be provided in the aqueous solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the equivalent circuit mode for B-DNA and M-DNA. The circuit within the dofted box is the standard Randles circuit. R_(s): solution resistance, R_(x): resistance through the DNA, R_(ct): charge transfer resistance, CPE: constant phase element. W: Warburg impedance

FIG. 2 is a schematic illustration of native DNA (B-DNA) and metal DNA (M-DNA) on a gold electrode surface. As illustrated, the Zn²⁺ ions may be thought of as binding to the outside of the M-DNA as well as being inserted into the helix.

FIG. 3 shows cyclic voltammograms for (a) bare gold and (b) 20 base pair duplex B-DNA assembled on gold electrode in 4 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1:1), 20 mM NaClO₄ and 20 mM Tris-ClO₄ buffer solution (pH 8.6). Scan rate, 50 mV/s.

FIG. 4 shows XPS spectra of (a) bare gold, (b) 20 base pair duplex B-DNA assembled on gold and (c) 20 base pair duplex M-DNA assembled on gold.

FIG. 5 shows Nyquist plots (Z_(im) vs Z_(re)) with 4 mM Fe(CN)₆ ^(3−/4−) (1:1) mixture as redox probe 20 mM Tris-ClO₄ and 20 mM NaClO₄ solution, applied potential 0.250 V vs. Ag/AgCl. In all cases the measured data points are shown as ◯ with the calculated fit to the Randles circuit as ----- or modified Randles circuit as —. (A) Bare gold electrode, (B) 20 base pair duplex B-DNA assembled on gold electrode, (C) 20 base pair duplex M-DNA assembled on gold electrode and (D) 20 base pair duplex B-DNA assembled on gold electrode with 0.4 mM Zn²⁺ at pH 7.0 (3) or with 0.4 mM Mg²⁺ at pH 8.6 (□).

FIG. 6 shows Nyquist plots in the absence of a redox probe for (A) 20 base pair duplex B-DNA assembled on gold (□) and (B) 20 base pair duplex M-DNA assembled on gold (▪). The experimental data were fit to the equivalent circuit shown.

FIG. 7 shows Nyquist plots with Fe(CN)₆ ^(3−/4−) as redox probe for 15 base pair duplex monolayers as B-DNA (□) or M-DNA (▪), 20 base pair duplex monolayers as B-DNA (◯) or M-DNA (●), and 30 base pair duplex monolayers as B-DNA (Δ) or M-DNA (▴). The data points were fit to the modified Randles circuit as described in the text.

FIG. 8 shows Nyquist plot for the Impedance measurements for B-DNA and M-DNA modified gold electrode in 5 mM Ru(NH₃)^(3+/2+), 20 mM Tris-ClO₄ buffer solution (pH, 8.6), applied potential −0.10V vs. Ag/AgCl.

FIG. 9 is a schematic showing DNA mismatches in duplexes attached to a surface, as discussed in Example 2.

FIG. 10 is a graph showing impedance spectra for a perfect duplex and one containing a middle mismatch under B-DNA and M-DNA conditions, as discussed in Example 2.

FIG. 11 shows Nyquist plots (−Z_(im) VS Z_(re)) of the 20 base pair complementary B-DNA (◯), middle mismatch B-DNA(□), complementary M-DNA(●)and middle mismatch M-DNA(▪) assembled on gold in 4 mM [Fe(CN)₆]_(3−/4−) (1:1) mixture as the redox probe in 20 mM Tris-ClO₄ and 20 mM NaClO₄ solution. Applied potential of 250 mV versus Ag/AgCl. [Zn_(II)]=0.4 mM; pH 8.6. In all cases the measured data points are shown as symbols with the calculated fit to the equivalent circuit as solid lines. Inset: The experimental data were fit to the equivalent circuit. Rs: solution resistance, Rx: monolayer pinhole/defect resistance, RCT: charge transfer resistance, CPE: constant phase element, W: Warburg impedance.

FIG. 12. a) Hybridization-dehybridization procedure. i) soaked in water:EtOH (60:40) bath at 60° C. for 10 minutes then rinsed with room temperature 20 mM Tris-ClO₄ buffer. ii) add target ss-DNA in SSC buffer and allow duplex formation to occur for 10 minutes at 37° C. followed by 3 hours at room temperature. b) Nyquist plot of fully hybridized “ideal” monolayer of 1:2 construct (∘), ss-DNA monolayer of 1 (□) after dehybridization procedure and rehybridized ds-DNA film of 1:2 (●) following the rehybridization procedure. The impedance spectra of the rehybridized film is different compared to that of the “ideal” 1:2 films putatively indicating the heterogeneity of the monolayer as a result of incomplete hybridization.

FIG. 13 shows Nyquist plots (−Z_(im) VS Z_(re)) of the rehybridized 20 base pair complementary B-DNA (∘), middle mismatch B-DNA(□), complementary M-DNA(●) and middle mismatch M-DNA(▪) assembled on gold with 4 mM Fe(CN)₆ ^(3−/4−) (1:1) mixture as the redox probe in 20 mM Tris-ClO₄ and 20 mM NaClO₄ solution. Applied potential of 250 mV versus Ag/AgCl. [Zn^(II)]=0.4 mM; pH 8.6. In all cases the measured data points are shown as symbols with the calculated fit to the equivalent circuit as solid lines.

FIG. 14 illustrates the determination of detection limits by monitoring the change in RCT between B-DNA and MDNA as a function of target single-stranded DNA concentration. Complementary DNA strands (□) and middle mismatch DNA strands (∘). Error bars are derived from a minimum of 5 electrodes.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention, impedance spectroscopy has been used to probe the electronic properties of B-and M-DNA self-assembled monolayers on gold electrodes.

By way of background, FIG. 1 illustrates an electrical circuit modelling the impedance of an electrode undergoing heterogeneous electron transfer through a self-assembled monolayer, which is usually described on the basis of the model developed by Randles (Randles, 1947). The equivalent electrical circuit (FIG. 1 in dotted box) consists of resistive and capacitance elements. R_(s) is the solution resistance, R_(ct) is the charge transfer resistance, C is the double-layer capacitance and W is the Warburg impedance due to mass transfer to the electrode. In general the Randles circuit provides a good model for the behaviour of alkanethiol monolayers. Of considerable interest, is the observation that monolayers of HMB (4′-hydroxy-4-mercaptobiphenyl) which contain a conjugated π system cannot be adequately described by the Randles circuit; but if an additional resistance is added in parallel (R_(x) in FIG. 1) then the spectra can be fit well (Janek et al., 1998).

As shown in FIG. 2, upon addition of Zn²⁺ to form M-DNA the ions are inserted into the DNA helix as well as binding to the phosphate backbone outside the helix. The conversion of B- to M-DNA gives rise to characteristic changes in the impedance spectra which was observed for 15, 20 and 30 base pair duplexes. It was found that the modified Randles circuit which includes R_(x), a resistance in parallel, may be used to give a good fit to the experimental data (FIG. 1). Under these conditions, M-DNA appears to decrease both R_(x) and R_(ct), and promote electron transfer through the monolayer.

Various aspects of the invention involve M-DNA, a form of conductive metal-containing oligonucleotide duplex. In alternative aspects of the invention, the conductive metal-containing oligonucleotide duplex may include a first nucleic acid strand and a second nucleic acid strand, the first and second nucleic acid strands including respective pluralities of nitrogen-containing aromatic bases covalently linked by a backbone. The nitrogen-containing aromatic bases of the first nucleic acid strand may be joined by hydrogen bonding to the nitrogen-containing aromatic bases of the second nucleic acid strand. The nitrogen-containing aromatic bases on the first and the second nucleic acid strands may form hydrogen-bonded base pairs in stacked arrangement along a length of the conductive metal-containing oligonucleotide duplex. The hydrogen-bonded base pairs may include an interchelated metal cation coordinated to a nitrogen atom in one of the nitrogen-containing aromatic bases.

The interchelated metal cation may include an interchelated divalent metal cation. The divalent metal cation may be selected from the group consisting of zinc, cobalt and nickel. Alternatively, the metal cation may be selected from the group consisting of the cations of Li, Be, Na, Mg, Al, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Cs, Ba, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, TI, Pb, Bi, Po, Fr, Ra, Ac, Th, Pa, U, Np and Pu.

The first and the second nucleic acid strands may include deoxyribonucleic acid and the nitrogen-containing aromatic bases may be selected from the group consisting of adenine, thymine, guanine and cytosine. The divalent metal cations may be substituted for imine protons of the nitrogen-containing aromatic bases, and the nitrogen-containing aromatic bases may be selected from the group consisting of thymine and guanine. At least one of the nitrogen-containing aromatic bases may include thymine, having an N3 nitrogen atom, and the divalent metal cation may be coordinated by the N3 nitrogen atom. Alternatively, at least one of the nitrogen-containing aromatic bases may include guanine, having an NI nitrogen atom, and the divalent metal cation may be coordinated by the NI nitrogen atom.

In various aspects of the invention, as disclosed in the following examples, DNA monolayers may be assembled on a gold surface and assessed by cyclic voltammetery (CV) or X-ray photoelectron spectroscopy (XPS). As shown in the examples, the CV spectra may provide good evidence for a densely-packed monolaryer with good blocking against Fe(CN)₆ ^(3−/4−), From the XPS, the film thickness may be estimated based on the exponential attenuation of the Au 4f signal, calculated in the examples to be 45 Å. (Pressprich et al., 1989). A 20 base pair duplex may be expected to have a length of about 70 Å so a measured thickness of 45 Å is for examples consistent with the DNA protruding from the surface at an angle of about 50°. In general duplex DNA attaches through the linker as compared to single-stranded DNA which can also attach through the bases (Herne and Tarlov, 1997). In the examples, the value of 162.4 eV for the S_(2p) peak is in good agreement with previous reports for alkylthiols indicating that the DNA is interacting with the surface through a S—Au bond (Ishida et al., 1999).

AC impedance spectroscopy is a known method to probe and model the interfacial characterization of electrodes (Bard and Faulkner, 2001). Data may for example be presented as Nyquist plots (Z_(im) vs Z_(re)) in which characteristic changes may be readily observed and interpreted. The complex impedance may be presented as the sum of the real, Z_(re) (ω)), and the imaginary, Z_(im) (ω) components that may originate mainly from the resistance and capacitance of the measured electrochemical system, respectively. As exemplified herein, the Nyquist plot for a bare electrode is a semicircle region lying on the Z_(re) axis followed by a straight line. The semicircle portion, measured at higher frequencies, putatively corresponds to direct electron transfer limited process, whereas the straight linear portion, observed at lower frequencies, putatively represents the diffusion controlled electron transfer process. The modification of the metallic surface with an organic layer may decrease the double layer capacitance and retard the interfacial electron transfer rates compared to a bare metal electrode (Finklea et al., 1993; Kharitonov et al., 2000).

In some embodiments, data analysis may require modeling the electrode kinetics with an equivalent circuit consisting of electrical components. For many monolayers the commonly accepted equivalent circuit is based on the Randles model, as shown in FIG. 1. However, in order to obtain a good fit to the data from the examples disclosed herein, a parallel interfacial resistance R_(x) was added to the equivalent circuit, nominally corresponding to electron transfer through the DNA. Evidence for a parallel interfacial resistance may for example be provided by impedance measurements without the Fe(CN)₆ ^(3−/4−), redox-active probe (see FIG. 6).

For many uncharged monolayers, different redox probes may give qualitatively similar results, presumably because the interaction between the probe and the monolayer is not electrostatic (Boubour and Lennox, 2000; Finklea, 1996; Finklea et al., 1993). DNA, however, is negatively-charged and therefore, positively-charged probes such as Ru(NH₃)₆ ^(3+/2+) may enter the monolayer whereas negatively-charged probes such as Fe(CN)6^(3−/4−) may not. These differences are for example reflected in the results shown in the examples herein, where R_(ct) with Ru(NH₃)₆ ^(3+/2+) is about 1 kΩ (FIG. 8), similar to that of a bare electrode, whereas with Fe(CN)₆ ^(3−/4−) and B-DNA the corresponding value is nearly 20 kΩ. Therefore, Ru(NH₃)₆ ^(3+/2+) is not a suitable probe for DNA since the charge transfer can essentially by-pass the monolayer.

The results disclosed in the examples herein illustrate that under certain conditions, M-DNA may be a better conductor than B-DNA since both R_(ct) and R_(x) are smaller for M-DNA. In the examples, the difference between R_(ct) for B- and M-DNA tends to increase with increasing length whereas the difference in R_(x) decreases with increasing length of the DNA duplex. In the examples, the DNA was not directly attached to the electrode so that R_(x) and R_(ct) both contain terms in series for electron transfer from the DNA through the linker to the electrode. In alternative embodiments, the DNA may be attached directly or with linkers of variable lengths to resolve the influence of the linker. In some embodiments, the interconversion of B- and M-DNA may provide systems wherein both Rx and Rct can be modulated with changes in metal ion or pH.

EXAMPLE 1

In an example of some aspects of the invention, described in more detail below, monolayers of thiol-labelled DNA duplexes of 15, 20, and 30 base pairs were assembled on gold electrodes. Electron transfer was investigated by electrochemical impedance spectroscopy with Fe(CN)₆ ^(3−/4−) as a redox probe. The spectra, in the form of Nyquist plots, were analysed with a modified Randle circuit which included an additional component in parallel, R_(x), for the resistance through the DNA. For native B-DNA R_(x) and R_(ct), the charge transfer resistance, both increase with increasing length. M-DNA was formed by the addition of Zn²⁺ at pH 8.6 and gave rise to characteristic changes in the Nyquist plots which were not observed upon addition of Mg²⁺ or at pH 7.0. R_(x) and R_(ct) also increased with increasing duplex length for M-DNA but both were significantly lower compared to B-DNA. Therefore, certain metal ions can modulate the electrochemical properties of DNA monolayers and electron transfer via the metal DNA film is faster than that of the native DNA film.

Chemicals

Potassium hexaferricyanide, potassium hexaferrocyanide, hexaamineruthenium (III) chloride hexaammineruthenium (II) chloride, were from Aldrich and were ACS reagent grade. Zn(ClO₄)₂, Mg(ClO₄)₂ and Tris-ClO₄ were purchased from Fluka Co. The standard buffer was 20 mM Tris-ClO₄ at either pH 8.7 or 7.0. Other chemicals were analytical grade. All solutions were prepared in Millipore filtered water.

DNA

The probe DNAs were synthesized and purified with standard DNA synthesis methods at the Plant Biotechnology Institute, Saskatoon. The oligonuocleotides base sequences are: 15-mer DNA, 5′-AAC TAC TGG GCC ATC—(CH₂)₃—S—S—(CH₂)₃—OH—3′, target complementary sequence 5′-GAT GGC CCA GTA GTT-3′. 20mer DNA, 5′-AAC TAC TGG GCC ATC GTG AC—(CH₂)₃—S—S—(CH₂)₃—OH—3′, target complementary sequence 5′-GTC ACG ATG GCC CAG TAG TT-3′, 30 mer DNA, 5′-GTG GCT AAC TAC GCA TTC CAC GAC CAA ATG—(CH₂)₃—S—S—(CH₂)₃—OH—3′, target complementary sequence 5′-CAT TTG GTC GTG GAA TGC GTA GTT AGC CAC-3′.

Electrode Preparation

Gold disk electrodes (geometric surface area 0.02 cm²) and Ag/AgCl reference electrodes were purchased from Bioanalytical Systems. Before use, the electrodes were carefully polished with a 0.05 μm alumina slurry and then cleaned in 0.1 M KOH solution for a few minutes and then wash in Millipore H₂O, twice. The electrodes were carefully investigated by microscopy to ensure that there were no obvious defects. Finally, electrochemical treatment was preformed in the cell described below, by cyclic scanning from potential −0.1 to +1.25 V vs. Ag/AgCl in 0.5M H₂SO₄ solution until a stable gold oxidation peak at 1.1 V vs. Ag/AgCl was obtained (Finklea, 1996).

Preparation of DNA Modified Gold Electrodes

DNA duplexes were prepared by adding 10 nmol of the disulphide-labeled DNA strands to 10 nmol of the complementary strands in 50 μl of 20 mM Tris-ClO₄ buffer pH 8.7 with 20 mM NaClO₄ for 2 hr at 20° C. The final double-stranded DNA concentration is about 100 μM. The freshly prepared gold electrodes were incubated with the DNA duplexes for 5 days in a sealed container. The electrodes were rinsed thoroughly with buffer solution (20 mM Tris-ClO₄ and 20 mM NaClO₄) and mounted into an electrochemical cell. B-DNA was converted to M-DNA by the addition of 0.4 mM ZnClO₄ for 2 hrs at pH 8.7.

X-Ray Photoelectron Spectroscopy

A Leybold MAX200 photoelectron spectrometer equipped with an Al-Koc radiation source (1486.6 eV) was used to collect photoemmission spectra. The base pressure during measurements was maintained less than 10⁻⁹ mbar in the analysis chamber. The take-off angle was 60°. The routine instrument calibration standard was the Au 4f_(7/2) peak (binding energy 84.0 eV).

Electrochemistry

A conventional three-electrode cell was used. All experiments were conducted at room temperature. The cell was enclosed in a grounded Faraday cage. The reference electrode was always isolated from the cell by a Luggin capillary containing the electrolyte. The salt-bridge reference electrode was used because of limiting Cl⁻ ion leakage for the normal Ag/AgCl reference electrode to the measurement system. The counter electrode was a platinum wire. Impedance spectroscopy was measured with a 1025 frequency response analyzer (FRA) interfaced to an EG&G 283 potentiostat/galvanostat via GPIB on a PC running Power Suite (Princeton Applied Research). Impedance was measured at the potential of 250 mV vs. Ag/AgCl, and was superimposed on a sinusoidal potential modulation of ±5 mV. The frequencies used for impedance measurements can range from 100 kHz to 100 mHz. The impedance data for the bare gold electrode, B-DNA and M-DNA modified gold electrode were analyzed using the ZSimpWin software (Princeton Applied Research). In all impedance spectra, symbols represent the experimental raw data, and the solid lines are the fitted curves.

Results

Assembly of the Monolayer

Native duplex B-DNA was assembled on the gold surface as described in Materials and Methods. The monolayer was characterized by cyclic voltammetery with 4 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1:1) mixture, as a redox probe. A typical scan is shown in FIG. 3; the bare gold electrode shows a characteristic quasi-reversible redox cycle with a peak separation of 158 mV. For the 20 base pair duplex assembled on the electrode, the peak current drops by over 95% and the separation between the oxidation and reduction peaks is increased indicating the presence of the DNA on the electrode and a reduced ability for electron transfer between the solution and the surface.

The gold surface was also analysed by X-ray photoelectron spectroscopy (XPS). As shown in FIG. 4, the intensity of the Au_(4f) peaks decreases upon attachment of the DNA (either B- or M-DNA) as expected for a modified surface (Kondo et al., 1998; Ishida et al., 1999). The S_(2p) (162.4 eV), P_(2p) (133 eV) and N_(1s) (400 eV) peaks are evident in the spectra of B- and M-DNA but are not present in the spectrum of the bare gold providing good evidence for the attachment of a disulphide-linked DNA to the surface. Of particular interest is the observation that that the N_(1s) and O_(1s) spectra for B- and M-DNA (after addition of Zn²⁺ at pH 8.7) are different. This is consistent with the zinc ions interacting with the DNA double helix and more specifically with the nitrogen and oxygen atoms of the base pairs (Lee et al., 1993; Aich et al., 1999).

Impedance Spectroscopy for B-DNA

Impedance measurements were performed in the presence of 4 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1:1) mixture, as the redox probe. FIG. 5A shows a Nyquist plot for the bare gold electrode which can be described as a semicircle near the origin at high frequencies followed by a linear tail with a slope of unity. Others have described similar curves and the data can be fit adequately by the Randles circuit of FIG. 1. The diameter of the semicircle is a measure of the charge transfer resistance, R_(ct). For the 20-mer B-DNA (FIG. 5B) R_(ct) increases considerably compared to the bare electrode since electron transfer to the electrode is reduced. However, the low frequency region is no longer linear and cannot be fit adequately by a simple Randles circuit. Impedance measurements of B-DNA (and M-DNA, see below) in the absence of a redox probe (FIG. 6) demonstrated non-linear behavior which is not expected for a simple insulator. However, the curves of FIG. 6 could be fit with a simple circuit consisting of a capacitor with a resistance in parallel. This result suggests the presence of an additional interfacial resistance, R_(x) which can be added in parallel to the Randles circuit (FIG. 1). As shown in FIG. 5B, the modified circuit gives an excellent fit to the experimental data in both the low and high frequency zones.

Formation of M-DNA

Upon addition of Zn²⁺ to the 20-mer B-DNA modified gold electrode at pH 8.7 to form M-DNA, the impedance spectrum changed in a distinctive pattern with a reduction in Z_(im) and Z_(re) at both high and low frequencies (FIG. 5C). Control experiments demonstrated that M-DNA formation was complete within 2 hours. Again only the modified Randles circuit gives a good fit to the experimental data. Also shown in FIG. 5D are impedance spectra for the DNA modified electrode in a pH 7.0 buffer with and without Zn²⁺ and at pH 8.7 with Mg²⁺. Under these conditions, M-DNA does not form and only small changes in the impedance spectra are observed. The calculated values for R_(s), R_(x), R_(ct), C and W are listed in Table 1. It is clear that there are significant decreases in R_(x) and R_(ct) upon formation of M-DNA which are not found upon addition of Mg²⁺ nor upon addition of Zn²⁺ at pH 7.0. TABLE 1 Table I. DNA with pH and ion type^(a) Bare B-DNA M-DNA B-DNA DNA with DNA with Element Gold pH 8.6 pH 8.6 PH 7.0 Zn pH 7.0 Mg pH 8.6 Rs/Ω 302 320 338 327 313 334 Rx/Ω 16160 12850 15560 14650 14880 C/μF 2.6 0.288 0.285 0.289 0.318 0.355 RCT/Ω 1229 18830 10009 16180 15010 15360 W/10⁻⁵Ω s^(−1/2) 27 3.9 8.2 1.9 1.8 2.0 ^(a)Values derived from the modified Randles circuit except for the bare Au electrode for which the data were fit to the unmodified Randles circuit. DNA Sequence Length

In order to provide further information concerning the elements in the suggested model, DNA duplexes of 15, 20, and 30 base pairs were used to modify the surface of the gold electrode. As shown in FIG. 7, all of the impedance spectra have the same characteristic shape and a fit to the modified Randles circuit is excellent. The calculated values for R_(s), R_(x), R_(ct), C and W are listed in Table 2. There are two distinct trends. First, R_(x) and R_(ct) increase with increasing length for both B- and M-DNA. Second, for any length of duplex R_(x) and R_(ct) for M-DNA is less than the corresponding value for B-DNA. W, the Warburg impedance which represents mass transfer to the electrode is more variable but in all cases is higher for the M-DNA duplexes. As expected R_(s), the solution resistance, is independent of duplex length and C, the double layer capacitance decreases with increasing length of the duplex. TABLE 2 DNA with different length sequences 15 mer 15 mer 20 mer 20 mer 30 mer 30 mer Element B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA Rs/Ω 322 334 320 338 319 330 Rx/Ω 12500 7681 16160 12850 17630 15760 C/μF 0.679 0.621 0.288 0.285 0.291 0.271 R_(CT)/Ω 7936 5326 18830 10009 26370 16720 W/10⁻⁵Ω s^(−1/2) 2.7 3.2 3.9 8.2 2.3 5.5 Ru(NH₃)₆ ^(3+/2+) redox probe

The redox probe used in this Example was Fe(CN)₆ ^(3−/4−) which is negatively-charged and, therefore, may be repelled by the phosphodiester backbone of the DNA. Ru(NH₃)₆ ^(3+/2+), on the other hand, may be expected to be able to penetrate the monolayer. Impedance spectroscopy was performed with Ru(NH₃)₆ ^(3+/2+) as a redox probe for the 20 base pair B- and M-DNA duplexes (FIG. 8). As shown in the inset, R_(ct) is then relatively small and there is relatively little difference between the spectra for B-DNA and M-DNA.

EXAMPLE 2

In the previous example, the impedance spectroscopy of self-assembled monolayers (SAMs) of B-DNA and M-DNA is described, and it is shown that each gave characteristic values of resistance (R) and capacitance (C) which were dependent on DNA length and metal ion concentration. This example illustrates that single base pair mismatches in the DNA also give rise to well-defined changes in the impedance spectra so that a mismatch can be reliably distinguished from a perfect duplex under certain conditions.

The DNA sequences and position of the mismatches are shown in FIG. 9, and in Table 3. TABLE 3 Perfect match DNA C₃-SS-C₃- 5′-GTC ACG ATG GCC CAG TAG TT-3′ 5′-AAC TAC TGG GCC ATC GTG AC-3′ Middle Mismatch C₃-SS-C₃- 5′-GTC ACG ATG GCC CAG TAG TT-3′ 5′-AAC TAC TGG GTC ATC GTG AC-3′ Top mismatch C₃-SS-C₃- 5′-GTC ACG ATG GCC CAG TAG TT-3′ 5′-ATC TAC TGG GCC ATC GTG AC-3′ Bottom Mismatch C₃-SS-C₃- 5′-GTC ACG ATG GCC CAG TAG TT-3′ 5′-AAC TAC TGG GCC ATC GTG CC-3′

Methods used in this example are as set out in Example No. 1, unless indicated otherwise.

Impedance spectra for a perfect duplex and one containing a middle mismatch under B-DNA and M-DNA conditions are shown in the FIG. 10. Each point represents a value for Z_(i) and Z_(r) measured at a particular AC frequency. The points at 0.1 Hz and 49 Hz are for example highlighted and it can be seen that the corresponding values of Z_(i) and Z_(r) are very different for a perfect duplex and a mismatch and for B-DNA and M-DNA.

From the impedance spectra as shown in FIG. 10, it is possible to calculate the values of R and C with precision (as described in Example No. 1) and to use these to distinguish between a perfect duplex and a mismatch.

In some embodiments, different electrodes may give different values of R and C. Accordingly, in some embodiments, mismatch detection may be carried out using a matched set of electrodes. In alternative embodiments, because the difference between Z values for B-DNA and M-DNA may be more consistent and less dependent on the electrode and the experimental conditions, Z values may be measured at two frequencies for both B-DNA and M-DNA. From such data, it is possible to distinguish between a perfect duplex and a mismatched duplex. For example, ΔL_(i) may be defined as the difference between Z; for B-DNA and M-DNA measured at low frequency (0.1 Hz) and ΔH_(r) may be defined as the difference between Z_(r) for B-DNA and M-DNA at high frequency (49 Hz). A Y factor may be defined as Y=ΔL_(i)×ΔL_(r)×ΔH_(i)×ΔH_(r). In some embodiments, the measured Y factor for a perfect duplex may for example be about 1000 and for a mismatch may be from about 1 to about 40.

In one embodiment, a device that may for example be used for measuring Y factors is provided. Such a device comprising an array of electrodes each one of which would be individually addressable. A probe, such as a 20-mer duplex probe may be attached by a thiolate linkage to each electrode and the duplex denatured to leave only an attached single-stranded probe. This procedure may provide a more consistent electrode surface compared to attaching a single-strand directly. The target nucleic acid may then be hybridized to the electrodes and impedance measurements taken at two frequencies. The electrodes may then be treated to allow conversion to M-DNA, for example by treating with 0.2 mM ZnClO₄, and the impedance measurements repeated. In such embodiments, a measured Y factor below about 100 may be taken as indicative of a mismatch; whereas a value above about 100 may be taken to indicate a perfect duplex.

In some embodiments, careful measurements may allow the position of the mismatch to be detected, localizing the mismatch for example to the top, middle or bottom of the duplex. In some embodiments, such as single nucleotide polymorphism (SNP) detection, a sample from a heterozygote may for example give an intermediate Y value.

In some embodiments, polycrystalline gold electrodes may be used. Alternatively, monocrystalline electrodes may be used, which may improve the discrimination and enhance the sensitivity of the system.

In alternative embodiments, it will be appreciated that the systems of the invention may be used as data storage and readout devices in which information is stored in the form of the molecular configuration of a nucleic acid on an electrode.

EXAMPLE 3

In this Example, detection of a single-nucleotide mismatch in an unlabeled duplex DNA by electrochemical methods is presented. Impedance spectroscopy is used to characterize a perfect duplex monolayer and three DNA monolayers differing in the position of the mismatch. The monolayers were assayed as B-DNA and after conversion to M-DNA. Modeling of the impedance data to an equivalent circuit provides parameters that are useful in discriminating the four monolayer configurations. The resistance to charge transfer, R_(CT), was lower for all duplexes after conversion to M-DNA. Surprisingly, R_(CT) was also found to decrease for duplexes containing a mismatch. However, R_(CT) was found to be diagnostic for mismatch detection. In particular, the difference in R_(CT) between B- and M-DNA (ΔdR_(CT)) decreased from 190(22) Ω·cm2 for a perfectly matched duplex to 95(20) Ω·cm2, 30(20) Ω·cm2 and 85(20) Ω·cm2 for a mismatch at the top, middle and bottom positions, respectively.

In an alternative aspect of the invention exemplified herein, a method is used to form loosely-packed single-stranded (ss)-DNA monolayers, by duplex dehybridization, that are able to rehybridize to target strands. Rehybridization efficiencies were in the range of 40-70%. Under incomplete hybridization conditions, the R_(CT) was the same for matched and mismatched duplexes under B-DNA conditions. However, ΔR_(CT) between B- and M-DNA, under incomplete hybridization, still provided a distinction. The ΔR_(CT) for a perfect duplex was 76(12) Ω·cm2, whereas a mismatch in the middle of the sequence yielded a ΔR_(CT) value of 30(15) Ω·cm². The detection limit was measured and the impedance methodology reliably detected single DNA base pair mismatches at concentrations as low as 100 pM.

Materials

5′-disulfide-labeled and unlabeled oligonucleotide strands were synthesized by standard phosphoamidate solid-phase DNA synthesis using a fully automated DNA synthesizer, purified by reversed-phase HPLC and then characterized by electrospray ionization mass spectrometry. The DNA sequences and position of the mismatches are shown in Table 4. TABLE 4 Lists DNA sequences used for monolayer film preparation in Example 3. Mismatched base pairs are indicated by the bold characters in the sequence. Con- Strand struct Sequence Label 1:2 HO-(CH2)6-SS-(CH2)6- 5′-GTC ACG ATG 1 GCC CAG TAG TT- 3′ 3′-CAG TGC TAC 2 CGG GTC ATC AA- 5′ 1:3 HO-(CH2)6-SS-(CH2)6- 5′-GTC ACG ATG 1 GCC CAG TAG TT- 3′ 3′-CAG TGC TAC 3 CGG GTC ATC TA- 5′ 1:4 HO-(CH2)6-SS-(CH2)6- 5′-GTC ACG ATG 1 GCC CAG TAG TT- 3′ 3′-CAG TGC TAC 4 CTG GTC ATC AA- 5′ 1:5 HO-(CH2)6-SS-(CH2)6- 5′-GTC ACG ATG 1 GCC CAG TAG TT- 3′ 3′-CCG TGC TAC 5 CGG GTC ATC AA- 5′ Monolayer Preparation

Freshly cleaned gold electrodes (BAS, 1.6 mm diameter) were incubated in 0.05 mM ss-DNA or ds-DNA B-DNA, 20 mM Tris-ClO₄ buffer solution (pH 8.6) for 5 days. Then the electrodes were washed with Tris-ClO₄ buffer and mounted into an electrochemical cell. Dehybridization and regeneration of the single-stranded probe electrode was achieved by denaturing the duplex DNA by soaking the electrode in a heated (60° C.) water:EtOH (60:40) bath for 10 minutes then rinsing in room temperature 20 mM Tris-ClO₄ buffer. Reproducible behavior was found for repeated measurements on different electrodes. Rehybridization was performed by exposing the ss-DNA selfassembled monolayer (SAM) to SSC buffer (300 mM NaCl, 30 mM sodium citrate, pH 7) heated to 37° C. in the presence of target DNA for 10 minutes and then was allowed to cool to room temperature for an additional 3 hrs. B-DNA was converted to M-DNA by the addition of 0.4 mM Zn(ClO₄)₂·6 H₂O for 2 hrs at pH 8.6.

The formation of the monolayer was assessed by standard blocking studies with [Fe(CN)₆]_(3−/4−), X-Ray photoelectron spectroscopy (XPS) and EIS. The blocking studies showed a decrease in peak current attributed to the reduced diffusion of the redox probe to the Au surface. The XPS data showed the presence of an Au-thiolate bond and a thickness of 44 Å for a 1:2 monolayer.

Electrochemical Measurements

A conventional three-electrode cell was used. All experiments were conducted at room temperature (22° C.). The cell was enclosed in a grounded Faraday cage. The reference electrode was constructed by sealing a Ag/AgCl wire into a glass tube with a solution of 3 M KCl that was capped with a Vycor tip. The counter electrode was a platinum wire. Impedance spectra were measured using an EG&G 1025 frequency response analyzer interfaced to an EG&G 283 potentiostat/galvanostat. The AC voltage amplitude was 5 mV and the voltage frequencies used for EIS measurements ranged from 100 kHz to 100 mHz. The applied potential was 250 mV vs. Ag/AgCl, (formal potential, E₀, of the redox probe [Fe(CN)₆]_(3−/4−). All measurements were repeated a minimum of 5 times with separate electrodes to obtain statistically meaningful results.

Results

Monolayers of fully matched B-DNA on gold were prepared from the oligonucleotide 1 and its fully matched complementary strand 2. In order to evaluate the effect of mismatches by EIS, 3 types of mismatched monolayers were prepared, each containing a single pyrimidine-pyrimidine mismatch in the complementary strand. Complementary mismatched strand 3 contains a mismatch in the second top basepair, resulting in a mismatch distal to the electrode surface. Complementary mismatched strand 4 contains a T instead of a G in position 11, giving a monolayer with the mismatch in the middle of the duplex. Complementary mismatched strand 5 possesses a C instead of an A in position 19, resulting in a mismatch proximal to the electrode surface. Mismatched B-DNA monolayers of 1:3, 1:4, and 1:5 were prepared in an analogous manner. Impedance measurements were carried out on all monolayers in 20 mM Tris-ClO₄ (pH 8.6) in the presence of 4 mM [Fe(CN)₆]_(3−/4−) (1:1) mixture, as the solution-based redox probe. The B-DNA monolayers were then converted to M-DNA monolayers by the addition of 0.4 mM Zn_(II) at pH 8.6 as described elsewhere herein. The impedance measurements were repeated under M-DNA conditions for all four monolayers. Typical impedance spectra, in the form of Nyquist plots, for B-DNA and MDNA monolayers of a perfectly matched duplex (1:2) and a duplex containing a mismatch in the middle of the helix (1:4) are shown in FIG. 11. Each point represents a value of Z_(im) and Z_(re) measured at a particular AC frequency. The spectra show a lower impedance for M-DNA than for B-DNA, as would be expected from previous observations.₄₀₋₄₄ More importantly, the presence of a mismatch in the DNA duplex decreases the impedance of B-DNA while increasing the impedance of M-DNA. In order to provide a rationale for this behavior, the impedance spectra of all DNA films were analyzed with a modified Randles equivalent circuit. The circuit drawing is shown in FIG. 11. The same model was used to fit all monolayers described in this Example. The fit of the equivalence circuit to the experimental values is given as a solid line. This treatment allows the interpretation of the impedance data in terms of electronic circuit components, which are listed for all monolayers of this Example in Table 5. The equivalent circuit contains five elements that are described below. TABLE 5 Circuit element values for the complementary DNA monolayer and the series of mismatch DNA monolayers of this Example. The values in parentheses represent the standard deviations from several electrode measurements (n = 5) not the non-linear curve fitting errors. Circuit 1:2 1:3 1:4 1:5 Element B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA Rs/Ω · cm² 5.8 6.0 4.9 4.8 6.5 5.8 5.3 6.1 (0.5) (0.6) (0.9) (0.7) (0.7) (0.6) (0.7) (0.8) Rx/Ω · cm² 300 245 357 319 351 323 312 255 (21) (18) (19) (16) (17) (16) (15) (12) CPE*/μF · cm² 15.0 15.7 12.8 10.9 42.1 38.0 25.0 23.1 (0.4) (0.5) (0.3) (0.2) (0.9) (0.6) (0.3) (0.2) R_(CT)/Ω · cm² 390 200 299 204 258 228 317 232 (20) (10) (15) (12) (14) (11) (18) (10) ΔR_(CT)/Ω · cm² 190(22) 95(19) 30(18) 85(20) W/10⁻⁵Ωs^(−1/2) 1.5 3.9 3.8 3.4 7.8 8.1 3.0 3.9 (0.3) (0.4) (0.6) (0.2) (0.4) (0.5) (0.3) (0.2) *CPE and associated units are interpreted as a capacitor with an exponential modifier >0.9.

A solution resistance term, Rs, remains constant at 5-6 Ω·cm² under identical conditions of supporting electrolyte concentration and temperature. The circuit contains a constant phase element (CPE) modeled as a non-ideal capacitor, to account for inhomogeneity on the electrode surface. The CPE may be interpreted as a capacitor in situations where the exponential modifier is greater than 0.9. This is the case for all monolayers presented in this Example. Monolayer composition and thickness are contributing factors to the CPE. The magnitude of the CPE for films of the matched duplex 1:2 and the two top and bottom mismatched duplexes 1:3 and 1:5 were in the range of 10-25 μF·cm⁻². However, for films of 1:4, B-DNA and M-DNA containing the middle mismatch, a significantly higher capacitance of about 40 (2) μF·cm⁻² was observed.

The Rx component of the equivalence circuit can putatively be attributed to pinholes in the monolayer structure. The value of Rx is similar for each of the B-DNA monolayers, indicating the number and size of the pinholes does not change between monolayers. However, Rx tends to decrease upon conversion to M-DNA. The Warburg impedance element, W, is dependent on the rate of diffusion of the [Fe(CN)₆]_(3−/4−) redox probe. The Warburg impedance is smallest for the perfect duplex in the B-DNA conformation suggesting that this is the most ordered monolayer, which offers the least access of the solution electrophore through the DNA monolayer.

The charge transfer resistance term, RCT, may be viewed as comprising resistance terms resulting from (a) transfer of the electron from the [Fe(CN)₆]_(3−/4−) redox probe to the DNA monolayer, (b) the resistance to charge transfer between the base pairs of the DNA helix and (c) from the helix to the surface of the gold electrode. For all monolayers, RCT is lower for M-DNA than B-DNA. RCT allows the discrimination between a single nucleotide mismatch and a perfectly-matched DNA film. The presence of a mismatch causes an unexpected decrease in RCT for all films containing mismatches in this Example. For mismatch detection, the evaluation of the difference in charge transfer resistance, ΔRCT, between B-DNA and M-DNA for a given film provides a means for discrimination between a perfect duplex and one containing a single mismatch at either the top or middle positions of the duplex. Table 5 lists the ΔRCT for all films. ΔRCT for the perfectly matched duplex film 1:2 is 190 (22) Ω·cm² whereas for the mismatched films, ΔRCT is significantly smaller. Interestingly, ΔRCT for the top mismatch containing film of 1:3 and the bottom mismatch (1:5) are similar (95(19) Ω·cm² for 1:3 and 85(20) Ω·cm² for 1:5). ΔRCT for the duplex containing the middle mismatch is much lower (30(18) Ω·cm² for 1:4). The use of ΔRCT may for example be advantageous in alternative embodiments because different electrode morphologies may yield different impedances in circumstances where the comparative impedance measurements between B-DNA and M-DNA are reproducible.

To illustrate mismatch determination under non-ideal conditions, the effect of rehybridization is exemplified herein. In this format, the DNA probe sequence was washed across a ss-DNA monolayer (which may result in differences in hybridization). The direct formation of a ss-DNA monolayer may yield a film in which the DNA strands are densely packed and may interfere with the binding of the complementary strand. Therefore, in this Example a ds-DNA film is formed and then dehybridized to a more loosely packed ss-DNA monolayer. In this way, rehybridization efficiencies for the target DNA in the range of 40-70% may be achieved in some embodiments. FIG. 12 a schematically illustrates the hybridization-dehybridization procedure. Washing of a ds-DNA film with hot (60° C.) water:EtOH (60:40) bath followed by rinsing in room temperature Tris-ClO₄ buffer results in dehybridization and formation of a ss-DNA film consisting of DNA strand 1. This film is then exposed to solutions of complementary target ss-DNA and allowed to hybridize for 3 hours. In some embodiments, the heating may have deleterious effects on the monolayer, however, this may be ruled out where, as in the present Example, the Rx component remains essentially the same or increases, putatively indicating that no new pinholes or defect sites were created. In the present Example, upon reformation of the ds-DNA film using 2 or 4 following the dehybridization-rehybridization procedure, the impedance signal does not return to the values for a perfect ds-DNA monolayer as shown by FIG. 12 b, suggesting that the resulting film may consist of ds-DNA and ss-DNA. Importantly, despite the apparently incomplete rehybridization, the presence of a mismatch can still be detected as shown by the impedance spectra in FIG. 13. After rehybridization of the ss-DNA film with a matching complementary strand, 2, and one containing a single mismatch in the middle of the strand, 4, all spectra were fit to the same equivalent circuit described above and the electronic circuit parameters are shown in Table 6. TABLE 6 Fitted impedance values for the rehybridized complementary DNA monolayer and the rehybridized middle mismatch DNA monolayer. The values in parentheses represent the standard deviations from several electrode measurements (n = 5) not the non-linear curve fitting errors. Rehybridized 1:2 Rehybridized 1:4 Element B-DNA M-DNA B-DNA M-DNA Rs/Ω · cm² 6.3 (0.6) 6.0 (0.5) 5.8 (0.6) 5.9 (0.8) Rx/Ω · cm² 345 (11) 311 (15) 355 (17) 334 (9) CPE*/μF · 26 (1.5) 17 (0.6) 34 (0.9) 20 (1.5) cm² R_(CT)/Ω · cm² 295 (11) 219 (5) 284 (12) 255 (9) ΔR_(CT)/Ω · cm² 76 (12) 29 (15) W/10⁻⁵Ω s^(−1/2) 3.1 (0.1) 4.9 (0.3) 3.9 (0.4) 5.2 (0.4) *CPE and associated units are interpreted as a capacitor with an exponential modifier >0.9.

Apparent from the embodiment of FIG. 13 and Table 6 is that the B-DNA films, which result from the hybridization of a matched and mismatched DNA target, may be indistinguishable under some conditions. However, in this embodiment the films clearly show a difference under M-DNA conditions. Again, RCT may be used to discriminate between matched and mismatched DNA films. The difference in RCT between B-DNA and M-DNA is consistently larger in the exemplified embodiment (76(12) Ω·cm²) for a perfect duplex compared to a mismatched film in which ΔRCT decreases to 29(15) Ω·cm².

Rehybridization embodiments are exemplified at various concentrations of target complementary strand, to illustrate the determination of a minimum concentration of target ss-DNA required to discriminate a matched film from a mismatched DNA film. Each time, the impedance spectra were recorded for B-DNA and M-DNA films and fit to the equivalent circuit. As shown in FIG. 14, ΔRCT remains relatively constant down to concentrations of 100 pM of target ss-DNA. As exemplified herein, a clear discrimination between matched and mismatched DNA may be obtained by the difference in RCT between B-DNA and M-DNA.

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CONCLUSION

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. 

1. A method for detecting base pair mismatches in a nucleic acid duplex tethered to an electrode in an electrochemical circuit, comprising: a) applying electrical energy to the electrode in the electrochemical circuit; b) collecting electrochemical circuit data related to the impedance of the nucleic acid duplex on the electrode in the circuit; and, c) fitting the electrochemical circuit data to a circuit model to obtain circuit performance information indicative of a base pair mismatch in the nucleic acid duplex.
 2. The method of claim 1, wherein collecting electrochemical circuit data comprises measuring impedance spectra.
 3. The method of claim 2, wherein the impedance spectra are measured in a frequency domain.
 4. The method of claim 1, wherein the electrochemical circuit data comprises a measure of complex impedance.
 5. The method of claim 1, wherein the electrical energy is applied in an impedance spectroscopy system, and the impedance spectroscopy system comprises applying a sinusoidal signal at a constant frequency and a constant amplitude within a discrete period.
 6. The method of claim 1, wherein the circuit model comprises as circuit elements: a solution resistance Rs; a charge transfer resistance RCT; a constant-phase element CPE; a mass transfer element W (Warburg impedance); and, a resistance in parallel Rx; and wherein the circuit elements are arranged as follows:


7. The method of claim 1, wherein the nucleic acid duplex is a deoxyribonucleic acid.
 8. The method of claim 1, wherein the nucleic acid duplex comprises a metal-containing nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, the first and the second nucleic acid strands comprising a plurality of nitrogen-containing aromatic bases covalently linked by a backbone, the nitrogen-containing aromatic bases of the first nucleic acid strand being joined by hydrogen bonding to the nitrogen-containing aromatic bases of the second nucleic acid strand, the nitrogen-containing aromatic bases on the first and the second nucleic acid strands forming hydrogen-bonded base pairs; in stacked arrangement along the length of the conductive metal-containing nucleic acid duplex, the hydrogen-bonded base pairs comprising an interchelated divalent metal cation coordinated to a nitrogen atom in one of the aromatic nitrogen containing aromatic bases.
 9. The method of claim 1, further comprising comparing the circuit performance information of a first nucleic acid duplex to the circuit performance information of a second nucleic acid duplex.
 10. The method of claim 9, wherein the first nucleic acid duplex is B-DNA and the second nucleic acid duplex is a metal-containing nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, the first and the second nucleic acid strands comprising a plurality of nitrogen-containing aromatic bases covalently linked by a backbone, the nitrogen-containing aromatic bases of the first nucleic acid strand being joined by hydrogen bonding to the nitrogen-containing aromatic bases of the second nucleic acid strand, the nitrogen-containing aromatic bases on the first and the second nucleic acid strands forming hydrogen-bonded base pairs in stacked arrangement along the length of the conductive metal-containing nucleic acid duplex, the hydrogen-bonded base pairs comprising an interchelated divalent metal cation coordinated to a nitrogen atom in one of the aromatic nitrogen-containing aromatic bases
 11. The method of claim 1, wherein the circuit performance information is plotted on a Nyquist plot.
 12. The method of claim 1, wherein a plurality of nucleic acids form a monolayer of nucleic acid duplexes on the electrode.
 13. The method of claim 1, wherein the electrochemical circuit comprises an aqueous electrolyte and the nucleic acid duplex is tethered and solvated in an aqueous electrolyte.
 14. The method of claim 13, further comprising a redox probe in an aqueous solution.
 15. The method of claim 1, wherein the nucleic acid duplex is an double helix.
 16. A system for detecting base pair mismatches in a nucleic acid duplex tethered to an electrode in an electrochemical circuit, the system comprising: a) means for applying electrical energy to the electrode in the electrochemical circuit; b) means for collecting electrochemical circuit data related to impedance of the nucleic acid duplex on the electrode in the circuit; and, c) means for fitting the electrochemical circuit data to a circuit model to obtain circuit performance information indicative of a base pair mismatch in the nucleic acid duplex.
 17. A system for detecting base pair mismatches in a nucleic acid duplex tethered to an electrode in an electrochemical circuit, the system comprising: a) an electrical current source for applying electrical energy to the electrode in the electrochemical circuit; b) a controller for collecting electrochemical circuit data related to impedance of the nucleic acid duplex on the electrode in the circuit; and c) an analyzer for fitting the electrochemical circuit data to a circuit model to obtain circuit performance information indicative of a base pair mismatch in the nucleic acid duplex.
 18. The system of claim 17, further comprising a display for displaying the circuit performance information.
 19. The system of claim 17, further comprising a recorder for recording the circuit performance information.
 20. (canceled) 